The Caliciviridae are a family of positive-strand RNA viruses and consist of four genera designated: (1) Norovirus (with species Norwalk virus) (2) Sapovirus (with species Sapporo virus); (3) Vesivirus (with species, feline calicivirus and vesicular exanthema of swine virus); and (4) Lagovirus (with species rabbit hemorrhagic disease virus and European brown hare syndrome virus). Norwalk virus (NV) is the prototype strain for the genus Norovirus and was discovered by LID researchers in 1972. The Noroviruses are the major cause of nonbacterial epidemic gastroenteritis that occurs in family, school, institutional, or community-wide outbreaks, affecting all age groups. The human Noroviruses are genetically diverse and cannot be grown in cell culture, which has been a continuing research obstacle. A major goal of this laboratory is the development of control strategies for the caliciviruses (predominantly the noroviruses) associated with gastroenteritis. In order to accomplish this goal, basic knowledge of the epidemiology, immunology, and replication strategies of these viruses is needed. This year, we continued our comparative studies of representative viruses from three distinct Caliciviridae genera- Norovirus, Sapovirus, and Vesivirus. Two viruses, feline calicivirus (Vesivirus) and porcine enteric calicivirus (Sapovirus) grow in cell culture, and we have developed both systems further to study replication in cells. Feline calicivirus grows efficiently without the addition of exogenous factors, but porcine enteric calicivirus requires the presence of intestinal content (IC) fluid from an uninfected pig in the cell culture medium. After several years of searching for the active factor in IC with our collaborator, Dr. Linda Saif, we have now identified the active factors in IC to be bile acids. Although the precise mechanism by which the bile acids allow the growth of porcine enteric calicivirus in cell culture is yet to be determined, we observed an association between the down-regulation of STAT-1 activation and growth of the virus in cells. It is possible that the suppression of the cell's innate immunity might help us to identify a permissive cell culture system for the human noroviruses, and such efforts are in progress. Since human norovirus strains such as Norwalk virus and MD145-12, cannot yet be grown in cell culture, we have focused on study of their nonstructural proteins in in vitro systems. This year, we discovered that the norovirus polymerase is enzymatically active when expressed in bacteria, and that it is active in two forms- the ProPol precursor and mature Pol. We also found that the FCV recombinant polymerase, ProPol, along with its cell culture system, has potential for use as a drug-screening platform for anti-calicivirus drugs. We also characterized the complete genome of a GII/4 norovirus that caused an outbreak in a Maryland hospital in February, 2004 in order to compare its relatedness to the predominant GII/4 norovirus strain that caused outbreaks of gastroenteritis in Maryland nursing homes in the late 1980's. We have identified differences in the genomes of these two viruses that could give insight into how the noroviruses change over time. Our collaborative efforts with Dr. Richard Lloyd at Baylor University resulted in a co-publication this year in which we show for the first time that caliciviruses are associated with a shut-off host cell translation and that the virus-encoded proteinase cleaves poly A binding protein, a critical component of the eukaryotic translation machinery. Our collaboration with Dr. Herbert (Skip) Virgin at Washington University in the characterization of a new murine norovirus has also proceeded well, and we have completed the proteolytic cleavage map of the virus-encoded nonstructural polyprotein.